Total Phenolic and Flavonoid Contents of Aqueous Extract of Stinging Nettle and In Vitro Antiproliferative Effect on Hela and BT-474 Cell Lines.

Phenolic compounds including flavonoids and phenolic acids are plants secondary metabolites. Due to their ability to act as antioxidant agents, there is a growing interest to use those components in traditional medicine for cancer prevention or treatment. The aim of this study was to measure the amounts of total phenolics and flavonoids as well as anti-proliferative effect of aqueous extract of Stinging nettle on BT-474 and Hela cell lines. The amounts of phenolics content and total flavonoids were determined by folin ciocalteu and aluminium chloride methods, respectively. The free radical scavenging activity was measured by using diphenyl - picrylhydrazyl (DPPH). The reducing power of the extract was measured in the presence of potassium hexacyanoferrate and its antiproliferative activity was assessed on BT-474 and Hela cell lines using MTT assay. Total phenolic content was 322.941± 11.811 mg gallic acid/g extract. Total flavonoid content was 133.916±12.006 mg Catechin/g. The IC50 of DPPH radical was 1.2 mg/ ml and the reducing power was 218.9± 15.582 μg ascorbic acid/ g. Cell viability of BT-474 cells decreased to less than half of the control (no added extract) at the presence of 3 mg/ ml extract while no significant changes were detected for Hela cells at similar conditions. There was no significant difference in the percentage of surviving cells between consecutive days (day 1, 2 and 3) for both BT-474 and Hela cells (P>0.05). Although the relatively high amount of phenolic and flavonoid contents of the aqueous extract make this plant a promising candidate for diseases treatment; however, there is not a direct relationship between the amounts of these antioxidant components and the efficiency in in vitro cancer treatment.

During cell metabolism pathways, all living cells generate free radicals as part of normal cellular functions (5). Free radicals are highly reactive and may be either oxygen derived or nitrogen derived and can react with proteins, lipids, carbohydrates and DNA (6). An imbalance between reactive oxygen species and antioxidants may cause oxidative stress, leading to cellular damage and subsequently various diseases in man such as atherosclerosis, diabetes mellitus, arthritis, ischemia heart disease, gastritis, immunosuppression, neurodegenerative diseases, ageing and cancer (7)(8)(9)(10). An inverse association between consumption of fruits and vegetables with lower risk of cancer is widely accepted and there have been great efforts in cancer treatment with their secondary metabolites (11)(12)(13)(14)(15)(16). Therefore, the objective of this research was to measure the amounts of total phenolics, total flavonoids and anti-proliferative effect of aqueous extract of Stinging nettle on BT-474 and Hela cell lines.

Plant extract preparation
An aqueous extract of leaves of Stinging nettle was obtained previously as described by Fattahi et al. (17). Briefly, 15 g of plant leaves were powdered and extracted with 300 ml of 5% ethanol by boiling for 15 min. After filtration, the filtrate was then evaporated and stored at-20 o C for further analysis.

Determination of total phenolics content
Folin Ciocalteu reagent was used for analysis of total phenolics content (18). Briefly, 0.5 ml of the extract was mixed with 0.5 ml of Folin-Ciocalteu reagent. The solution was kept at 25 o C for 5-8 min before adding 2 ml of sodium carbonate solution 7.5 % and adjusting the volume to 8 ml with water. After 2 h, the absorbance was measured at 725 nm. Gallic acid was used as standard for the calibration curve. Total phenolic content was expressed as mg gallic acid equivalents per gram of sample (mg/g).

Determination of total flavonoids content
The total flavonoid content was measured by a colorimetric assay (19). One hundred micro liters of extract was added to 4 ml of distilled water. Then, 0.3 ml 5% sodium nitrite was added. After 5 min, 0.3 ml of 10% aluminium chloride was added. In 6 min, 2 ml of 1 M sodium hydroxide was added to the mixture. Immediately, the mixture was diluted by the addition of 3.3 ml distilled water and mixed thoroughly. The absorbance was determined at 510 nm versus a blank. Catechin was used as standard for the calibration curve. Total flavonoids content of the extract was expressed as mg catechin equivalents per gram of sample (mg/g).

DPPH radical scavenging activity
The free radical scavenging activity of Stinging nettle was measured by using diphenylpicrylhydrazyl (DPPH) assay. 5 ml of 80 mM DPPH radical solution was added to 1 ml of the Stinging nettle extract solutions ranging from 0.375 to 3 mg/ ml. The reaction was allowed for 30 min and absorbance was measured at 515 nm using a spectrophotometer (Rayto, China). IC50 value, the concentration of sample required to scavenge 50% of DPPH free radical, was calculated from the plotted graph of radical scavenging activity against the concentration of extracts.

Reducing power assay
The reducing power assay of the extract was measured according to the method used by mixture and centrifuged at 3000 rpm for 10 min.
2.5 ml of supernatant was mixed with 2.5 ml of distilled water and 0.5 ml of FeCl3 (0.1%) and the absorbance was measured at 700 nm. The results were compared with ascorbic acid which was used as a positive control. Reducing power assay of the extract was expressed as µg ascorbic acid equivalents per gram of sample (µg/g).

Cell lines
The Hela and BT-474 cell lines were purchased from Pasteur institute, Tehran, Iran. The cell lines were cultured in RPMI-1640 medium (PAA, Austria) supplemented with 10%fetal bovine serum and 1% antibiotics (penicillin/ streptomycin (Invitrogen)) in a humidified atmosphere containing 5% CO2 and 95% air, at 37°C.

Total phenolics content
Total phenolic content was estimated by gallic acid (Figure 1) and expressed as mg gallic acid equivalent (GAE)/g of extract. Table 1 Table 1.   Previous reports indicated a direct association between the amount of polyphenols present in fruits and vegetables and their cancer prevention ability (11)(12)(13). In this study we investigated the amount of total phenolics and flavonoids contents and radical